A laboratory scale semi-batch fed anaerobic ammonia oxidation (ANAMMOX) reactor was operated in the lab under two different feeding operations. In the first scenario, termed as phase I, the reactor was seeded and operated with NO2-N added externally with the filtrate to the reactor in the ratio needed for the successful ANAMMOX. A second reactor was also initiated shortly after the start-up of the ANAMMOX to accomplish partial nitrification (nitritation reactor) to generate NO2-N. In phase II, the operation of the ANAMMOX reactor was switched to the mode in which case the partially nitrified effluent from the nitritation reactor was fed to the ANAMMOX reactor. In both phases, real filtrate from a local wastewater treatment plant was used as the feed. The ANAMMOX reactor sustained a loading rate (average 0.33 ± 0.03 with a max of 0.4 g N (L day)−1) which is comparable with many other fed-batch reactors in the literature. Consistent total N removal (average of 82 ± 4%) could be sustained in the ANAMMOX reactor during both phases. The nitritation reactor also consistently enabled a NO2-N to NH3-N ratio of 1.2:1 which was needed for the successful operation of the ANAMMOX reactor in phase II. Sequence analysis and FISH showed that Kuenenia stuttgartiensis dominated the enriched ANAMMOX community along with several unidentified, but seemingly enriched, potential ANAMMOX strains. Microbial ecology analysis for nitritation reactor showed the dominance of Nitrosomonas europaea. In summary, this manuscript provides important information on the start-up and operation of anammox reactor with detailed investigation on microbial ecology in this reactor. Biotechnol. Bioeng. 2013; 110: 1180–1192. © 2012 Wiley Periodicals, Inc.